The conjugation of synthetic molecules to proteins is a key strategy in biomedical research, allowing for proteins to be immobilized onto surfaces, modified with chromophores to prepare in vitro sensors, or tagged with in vivo imaging agents (Wu et al., 1997. Fluorescence Spectroscopy 278:321-330; Harris et al, 2003, Nat. Rev. Drug Disc. 2:214-221; Wang et al 2005, Angew. Chem. Int. Ed. 44:34-66; Basle et al., 2010, Chem. Biol. 17:213-227; Canalle et al., 2010, Chem. Soc. Rev, 39:329-353). Examples of such conjugation include protein microarrays to investigate protein-protein interactions, and proteins labeled with near-IR quantum dots for imaging in deep tissue. Protein modification can also significantly improve protein properties so that they may be used as therapeutic and diagnostic agents: thermostability, resistance to degradation (both enzymatic and non-enzymatic in nature), increase in solubility, and improved formulation characteristics. Degradation-resistant modifications have been achieved by PEG modification of proteins and diagnostic antibodies. In most cases, the formation of well-defined conjugates is valuable, if not essential, for product development.
The protein termini are attractive targets for conjugation, because in many cases appending synthetic molecules at the termini will have minimal undesired effects on protein folding and function, especially when the terminus is relatively unstructured. (Alouani et al., 1995, Eur. J. Biochem. 227:328-334; Kinstler et al., 1996, J. Pharm. Res. 13; 996-1002; Gale et al., 1999, J. Chem. Soc. Perkin 1:2267-2270; Ramachandiran et al., 2000, J. Biol. Chem. 275:1781-1786; Jones et al., 2001, J. Bioconj. Chem. 12:1012-1020; Kinstler et al., 2002, Adv. Drug Deliv. Rev. 54:477-485; Chelius et al., 2003, Bioconj. Chem. 14:205-211; Arduini et al., 2004, Protein Exp. Purif. 34; 229-242; Mamaev et al., 2004, J. Anal. Biochem. 326:25-32; Baker et al., 2006, Bioconj. Chem. 17:179-188; Scheck et al., 2007, ACS Chem. Biol. 2:247-251; Merkel et al., 2008, ChemBioChem 9:1220-1224; Sharon et al., 2008, Biomaterials 29:3137-3142; Ebhardt et al., 2009, Anal. Chem. 81:1937-1943; Gao et al., 2009, Proc. Natl. Acad. Sci. USA 106:15231-15236; Sayers et al., 2009, Soft Matter 5; 3038-3046; Xiao et al., 2009, Org. Lett. 11:4144-4147; Jia et al., 2010, Mol. Biosyst. 6:1829-1833; Wang, et al., 2010, Anal. Biochem. 412:114-11.6; Wildes, et al., 2010, Proc. Natl. Acad. Sci. USA 107:4561-4566; Wu et al., 2010, J. App. Poly. Sci. 118:3269-3273). In addition, functionalized N-termini can be ligated to synthetic peptides to produce semi-synthetic proteins (Hackenberger et al., 2008, Angew. Chem. Int. Ed. 47:10030-10074).
Selective N-terminal modification has been achieved by various chemical and enzymatic methods, each with benefits and drawbacks. Small-molecule strategies permit the attachment of molecules to the protein N-terminus, but are subject to side reactions, incomplete specificity for the N-terminus and may need to be carried out in organic solvent mixtures (Scheck et al., 2007, ACS Chem. Biol. 2:247-251; Dixon et al., 1962, Biochem. J. 84:462-468; Dixon et al., 1964, Biochem. J. 92; 661-666; Geoghegan et al., 1979, Biochemistry 18; 5392-5399; Dixon, 1984, J. Protein Chem. 3:99-108; Acharya et al., 1987, Biochemistry 26:3524-3530; Qasmi et al., 1994, Tet. Lett. 35:4343-4344; Li et al., 2000, Tet. Lett. 41:4069-4073; Gilmore et al., 2006, Angew. Chem. Int. Ed, 45:5307-5311; Scheck et al., 2008, J. Am. Chem. Soc. 130:11762-11770; Witus et al., 2010, J. Am. Chem. Soc. 132; 16812-16817). Reverse proteolysis methods can be used to modify the N-terminus under conditions that do not require protein unfolding, but the reaction can be difficult to drive to completion in the absence of high protein concentrations (Kuhl et al., 1980, Tet. Lett. 21:893-896; Jakubke et al., 1985, Angew. Chem. Int. Ed. 24:85-93; Schellenberger et 1991, Angew. Chem. Int. Ed. 301437-1449; Chang et al., 1994, Proc. Natl. Acad. Sci. USA 91:12544-12548; Jackson et al., 1994, Science 266:243-247; Bordusa et 1997, Angew. Chem. Int. Ed. 36:2473-2475; Braisted et 1997, Solid-Phase Peptide Synthesis 289:298-313; Atwell et al., 1999, Proc. Natl. Acad. Sci. USA 96:9497-9502; Bordusa, 2002, Chem. Rev. 102:4817-4867; Tolbert et al., 2002, Angew. Chem. Int. Ed. 41:2171-2174; Muir, 2003, Annu. Rev. Biochem. 72:249-289; Wehofsky et al., 2003, J. Am. Chem. Soc. 125:6126-6133; Gentle et al., 2004, Bioconj. Chem. 15:658-663; Yoshihara et al., 2008, Bioorg. Med. Chem. Lett. 18; 6000-6003), Chemoenzymatic methods have been shown to function under mild conditions but require that moderate-sized target sequences be appended to the protein (Mazmanian et al., 1999, Science 285; 760-763; Mao et al., 2004, J. Am. Chem. Soc. 126:2670-2671; Tanaka et al., 2005, FEBS Lett. 579:2092-2096; Popp et al., 2007, Nat. Chem. Biol. 3; 707-708; Fontana et al., 2008, Adv. Drug Deliv. Rev. 60:13-28; Heal et al., 2008, Org. Biomol. Chem. 6:2308-2315; Tsukiji et al., 2009, ChemBioChem 10:787-798; Nelson et al., 2010, ACS Chem. Biol. 5:1147-1155; Heal et al, 2012, Nat. Methods 7:105-117).
Aminoacyl tRNA transferases (such as E. coli AaT and V. vulnificus BpT) are members of a growing class of enzymes that use aminoacyl tRNAs in secondary metabolism (Lahoud et al., 2010, Nat. Chem. Biol. 6:795-796). AaT catalyzes the transfer of Leu, Phe, or Met from an aminoacyl tRNA to a protein bearing an N-terminal Arg or Lys (Kaji et al., 1963, Biochem. Biophys. Res. Comm. 10; 406-409; Suto et al., 2006, EMBO J. 25; 5942-5950; Watanabe et al., 2007, Nature 449:867-871). BpT catalyzes the transfer of Leu from an aminoacyl tRNA to a protein bearing an N-terminal Asp or Glu (Graciet et al., 2006, Proc. Natl. Acad. Sci. USA 103:3078-3083). The addition of Leu or Phe targets that protein for degradation by ClpA as part of the N-end rule pathway (Mogk et al., 2007, Trends Cell Biol. 17; 165-172; Tobias et al., 1991, Science 254; 1374-1377; Varshaysky, 2008, Nat. Struct. Mol. 15:1238-1240; Schuenemann et al., 2009, EMBO Rep. 10:508-514). Kaji and co-workers observed AaT aminoacylation activity in crude E. coli preparations (Kaji et al., 1963, Biochem. Res. Commun. 10:406-409; Kaji et al., 1965, J. Biol. Chem. 240:1185-1191; Kaji et al., 1965, J. Biol. Chem. 240:1192-1197). Soffer and Leibowitz subsequently reconstituted the purified AaT enzyme and characterized its specificity for both the RNA and amino acid components of the donor molecule, demonstrating its use in transferring a non-natural amino acid, p-fluorophenylalanine (Leibowitz et al., 1969, Biochem. Biophys. Res. Commun. 36:47-53; Leibowitz, et al, 1971, J. Biol. Chem. 246; 5207-5212; FIG. 1A). Graciet and co-workers showed that BpT activity could also be functionally reconstituted in vitro (Graciet et al., 2006, Proc. Natl. Acad. Sci. USA 103:3078-3083).
Recently, purified AaT has been used to modify proteins in vitro with a variety of non-natural amino acids charged onto tRNAs, either by chemical semisynthesis or the use of a mutant aminoacyl tRNA synthetase (aaRS) (Ebhardt et al., 2009, Anal. Chem. 81:1937-1943; Taki et al., 2006, ChemBioChem 7:1676-1679; Taki et al., 2007, Biopolymers 88:263-271; Taki et al., 2008, ChemBioChem 9:719-722; Ebisu et al., 2009, ChemBioChem 10:2460-2464). Abramochkin and Shrader discovered that AaT was tolerant of variation in the acceptor stem (aminoacylation site) of the tRNA, and Sisido and co-workers showed that much shorter oligonucleotides can act as donors (Taki et 2008, ChemBioChem 9:719-722; Abramochkin et al., 1995, J. Biol. Chem. 270:20621-20628; Abramochkin et al., 1996, J. Biol. Chem. 271:22901-22907; for example in FIG. 1B, Rn=2-22 nucleotides). As part of their structural characterization of AaT, Tomita and co-workers found that AaT could bind phenylalanyl adenosine and transfer Phe to peptides in trace amounts (Watanabe et al, 2007, Nature 449:867-871). However, the use of adenosine mononucleoside as a donor substrate for AaT has not been further explored. No such exploration of donor scope has occurred for BpT.
There is thus a need in the art for methods of N-terminal modification that can be carried out easily under conditions that maintain protein folding without substantial prior protein manipulation. There is a further need in the art for methods of performing protein ligation, including ligation methods that are traceless in nature. The present invention addresses this unmet need in the art.